Differential regulation of ferritin subunits and iron transport proteins: an effect of targeted hepatic X-irradiation

Abstract

The current study aimed to investigate radiation-induced regulation of iron proteins including ferritin subunits in rats. Rat livers were selectively irradiated in vivo at 25 Gy. This dose can be used to model radiation effects to the liver without inducing overt radiation-induced liver disease. Sham-irradiated rats served as controls. Isolated hepatocytes were irradiated at 8 Gy. Ferritin light polypeptide (FTL) was detectable in the serum of sham-irradiated rats with an increase after irradiation. Liver irradiation increased hepatic protein expression of both ferritin subunits. A rather early increase (3 h) was observed for hepatic TfR1 and Fpn-1 followed by a decrease at 12 h. The increase in TfR2 persisted over the observed time. Parallel to the elevation of AST levels, a significant increase (24 h) in hepatic iron content was measured. Complete blood count analysis showed a significant decrease in leukocyte number with an early increase in neutrophil granulocytes and a decrease in lymphocytes. In vitro, a significant increase in ferritin subunits at mRNA level was detected after irradiation which was further induced with a combination treatment of irradiation and acute phase cytokine. Irradiation can directly alter the expression of ferritin subunits and this response can be strongly influenced by radiation-induced proinflammatory cytokines. FTL can be used as a serum marker for early phase radiation-induced liver damage.

Figure 1

(a) Western blot analysis of iron storage (FTL and FTH) and iron transport protein (TfR1, TfR2 and Fpn-1) in rat liver of control and irradiated animals. (b) Western blot analysis of iron storage (FTL and FTH) proteins from serum total protein. β-actin was used as a loading control in liver while, in serum loading control represents an internal loading control (∼55 kDa). Results are representative of three experiments.

Figure 2

(a) Changes of circulating AST levels after targeted hepatic irradiation. (b) Changes in hepatic iron content after single dose liver irradiation. Iron levels were determined by ferrozine-based assay (Co. control). Results represent mean values ± SEM (*P < 0.05 analyzed by one-way ANOVA; n = 3).

Figure 3

Changes in complete blood picture (CBC) after single dose liver irradiation. (a) Total leukocytes number. (b) Fold change in numbers of neutrophils and lymphocytes after targeted hepatic irradiation. Results represent mean values ± SEM (*P < 0.05 analyzed by one-way ANOVA; n = 3).

Figure 4

qRT-PCR analysis of total RNA from isolated rat hepatocytes treated with irradiation 8 Gy, alone or in combination with major acute phase cytokines (IL-6, IL-1β and TNF-α). Data are shown as fold changes in mRNA expression of iron storage proteins (FTL and FTH) at various time points relative to untreated controls for each time point. qRT-PCR was normalized by using two housekeeping genes: β-actin and ubiquitin C. Results represent means ± SEM of three experiments; *P < 0.05, n = 3.